Heterogeneous endotoxin detection bioassay using drug–nanoparticle bioconjugates: an optimization study

We present a detailed study of an endotoxin detection bioassay performed in a sandwich format on solid substrates. Self-assembled monolayers (SAMs) of C-18 alkyl silanes were immobilized on glass to heterogeneously entrap lipopolysaccharides (LPS) from human serum. The captured endotoxin was then tagged with polymyxin B sulfate–gold nanoparticle (GNP) conjugates followed by silver enhancement. The intensity of the obtained color spots were quantified using a mobile phone camera and the assay performance was optimized by studying the effects of various experimental parameters like silane concentration, temperature, humidity and incubation time. The assay gave a linear dynamic range of 10⁴ with a lower limit of detection (LOD) at 1 pg mL⁻¹. This LOD was further improved by an LPS pre-enrichment step prior to the assay. Rudimentary thermodynamic and kinetic models fitted to our experimental data suggest that LPS binding is an entropic process and LPS/GNP binding is mainly adsorption-controlled.